Search PubMed⌕ Search

Biomedical subjects

N Hilschmann

Publications and source records attributed to N Hilschmann.

At least 55 records · Page 3Linked to original sources
PubMed1991

Primary structure of the murine monoclonal IgG2a antibody mAb735 against alpha(2-8) polysialic acid. 1) Amino-acid sequence of the light (L-) chain, kappa-isotype.

Here we report the complete amino-acid sequence of the anti-alpha(2-8)polysialic acid antibody mAb735 light chain. The sequence was determined after digestion of the reduced and carboxymethylated L-chain with trypsin, SV-8 proteinase, and Asp-N proteinase, isolation of the generated peptides by RP-HPLC and characterization of these fragments by sequence analysis, amino-acid analysis and/or plasma desorption mass spectrometry. According to Kabat et al. the variable region belongs to the V kappa-II subgroup, whilst according to Hum et al. it belongs to the V kappa-1B subgroup. With the exception of proline at position 46, the sequence from position 1 to 95 is identical to the translated DNA sequence of a V kappa-germline gene segment previously reported.

Amino Acid Sequence↗
PubMed1991

Studies on human porin. VI. Production and characterization of eight monoclonal mouse antibodies against the human VDAC "Porin 31HL" and their application for histotopological studies in human skeletal muscle.

We report on the production and characterization of eight monoclonal mouse antibodies against the complete human VDAC "Porin 31HL". The antigen used was purified from a total membrane preparation of the transformed human B-lymphocyte cell line H2LCL. In Western blots all eight mAbs react with a single 31-kDa band in solubilized H2LCL membrane preparations thus demonstrating their specificity for the human VDAC "Porin 31HL". Concerning the epitope specificity we show that all eight mAbs equally react with the N-terminal part of human porin. Moreover, we demonstrate the expression of VDAC in the sarcolemma by indirect immunoenzyme labelling of cryosections of human skeletal muscle applying four of our mAbs. These data support our recent observations on the expression of porin channels in the plasmalemma of different normal and transformed human cell lines. VDAC in the plasmalemma is discussed as the molecular basis of the Blatz and Magleby channel.

Animals↗
PubMed1991

Studies on human porin. IV. The primary structures of "Porin 31HM" purified from human skeletal muscle membranes and of "Porin 31HL" derived from human B lymphocyte membranes are identical.

We report on the purification of "Porin 31HM" from the crude plasma membrane fraction of human skeletal muscle. Furthermore, all tryptic peptides of the molecule were purified and characterized by different methods. The alignment of the peptides with the complete primary structure of the human B lymphocyte plasma membrane-derived "Porin 31HL", published by us recently (Kayser, H. et al. (1989) this Journal 370, 1265-1278), proved both structures to be completely identical. Our data demonstrate that porin fractions from crude plasma membranes of different human cell types do not show any variation on the primary structure level.

Amino Acid Sequence↗
PubMed1991

The complete amino-acid sequence of the heavy chain of the human myeloma protein WIE, an immunoglobulin D.

The human myeloma protein WIE is a lambda-type immunoglobulin D; the amino-acid sequence of its Fc part and aminoethylated heavy chain was completely determined. The VH-part (subgroup III) begins N-terminally with 5-oxoproline, and it contains a long, unique CDR3 region. Since the constant part differs from known delta chains by one amino-acid substitution in the hinge region, IgD WIE probably represents an allotypic variant. As in other protein delta chains, O-glycosylations are confined to the hinge region. Furthermore, the ratios of N-glycosylations at the three positions are identical in IgD WAH [Takahashi, N. et al. (1984) J. Chromatogr. 317, 11-26.] and IgD WIE (100%, 50%, 100%). From the most conserved constant domain, C delta 3, a three-dimensional model was constructed to clarify the role of its delta-specific substitutions and glycosylation.

Amino Acid Sequence↗
PubMed1991

[Studies on human porin. V. The expression of "porin31HL" in the plasmalemma is not by cell transformation].

In recent papers we proved "Porin 31HL" to be located on the surface of human, EBV-transformed B lymphocytes. Here we present proof of "Porin 31HL" in the plasmalemma of normal human blood lymphocytes. For this purpose B and T lymphocytes were isolated from human heparinized blood and examined by indirect immunofluorescence techniques using different monoclonal antibodies against purified "Porin 31HL" and some B and T cell markers, respectively. For comparison a number of established cell lines of different origin were employed. Hence it followed that normal B and T cells as well as transformed and leukemic cells express "Porin 31HL" in their membrane. No significant quantitative differences could be seen. Consequently, the location of "Porin 31HL" in the plasmalemma is not a product of transformation.

Animals↗
PubMed1990

Studies on human porin. III. Does the voltage-dependent anion channel "Porin 31HL" form part of the chloride channel complex, which is observed in different cells and thought to be affected in cystic fibrosis?

"Porin 31HL", of known primary structure, is an integral protein of the plasmalemma of human B cells (Thinnes, F.P. et al. (1989) This Journal 370, 1253-1264; Kayser, H. et al. (1989) This Journal 370, 1265-1278). Purified "Porin 31HL" from human B lymphocytes was reconstituted into lipid bilayer membranes, where it formed defined voltage-dependent channels. Five minutes preincubation with 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, potent inhibitor of chloride transport, altered the channel-forming properties of the protein, so that it now showed small irregular channels instead of distinct steps. In addition, the voltage-dependence of the channel was abolished by the action of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate. Functional and structural similarities between "Porin 31HL" and porin preparations from other human tissues and from other species suggest that this protein may be part of the chloride channel complex, which is defective in cystic fibrosis.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1989

Gas-phase sequencing after electroblotting on polyvinylidene difluoride membranes assigns correct molecular weights to myoglobin molecular weight markers.

Commercially available polypeptide marker kits containing peptides generated by cyanogen bromide cleavage of either horse heart myoglobin or sperm whale myoglobin have been investigated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), followed by electroblotting on polyvinylidene difluoride membranes, and gas-phase sequencing. It could be shown that the molecular weights assigned to the SDS-PAGE bands by the companies are incorrect. Arranged in descending order, the marker kits are composed of the following polypeptide fragments from myoglobin: positions 1-153, 1-131, 56-153, 56-131, 1-55, and 132-153. A polypeptide comprising residues 1-14 was not found. According to these results the log Mr versus Rf plot used for calibration must be revised. For the separation of low molecular weight polypeptides and peptides a new gel system based on the theory of multiphasic zone electrophoresis combined with a modified Coomassie staining procedure is reported.

Amino Acid Sequence↗
PubMed1989

[Identification of human porins. I. Purification of a porin from human B-lymphocytes (Porin 31HL) and the topochemical proof of its expression on the plasmalemma of the progenitor cell].

We describe for the first time a porin (Porin 31HL) on the plasmalemm of an eukaryontic cell line, where porins have been found only on the outer mitochondrial membranes. The expression of the porin on the plasmalemm of transformed human B-lymphocytes is demonstrated by cytotoxicity- and indirect immunofluorescence techniques with living and fixed cells. The rabbit xenoantisera used were directed against purified Porin 31HL and free or acetylated synthetic peptides of its nineteen N-terminal amino acids. The three-step purification procedure for Porin 31HL started from a total membrane fraction of the B-cell line, followed by ion-exchange chromatography on CM- and DEAE-cellulose and a final gel filtration in SDS on Sephacryl S-300.

Amino Acids↗
PubMed1989

[Identification of human porins. II. Characterization and primary structure of a 31-lDa porin from human B lymphocytes (Porin 31HL)].

We characterize and describe for the first time the primary structure of a human porin with the molecular mass of 31 kDa derived from the plasmalemm of B-lymphocytes (Porin 31HL). Porin 31HL is shown to be a basic, channel forming membrane protein. The protein chain is composed of 282 amino acids with a relative molecular mass of 30641 Da without derivatisation. It is not a glycoprotein. The N-terminus is acetylated. Altogether the amino-acid sequence shows 56% hydrophilic or charged amino acids arranged in alternating regions of hydrophilic or hydrophobic character as it is typical for porins. In addition the 18 N-terminal amino acids of Porin 31HL can be arranged to an amphilic alpha-helix like in other porins. Porin 31HL shows approx. 29% or 24% identity to the primary structure of mitochondrial porins of Neurospora crassa and Saccharomyces cerevisiae. Partial data on mitochondrial porins from rat kidney and beef heart show sequence identity of about 90% to the human B cell porin elaborated here.

Amino Acid Sequence↗
PubMed1989

[The primary structure of crystallizable monoclonal immunoglobulin IgG1 Kol. II. Amino acid sequence of the L-chain, gamma-type, subgroup I].

The immunoglobulin Kol was the first intact antibody molecule which was characterized by high-resolution X-ray crystallography. Furthermore the complete amino-acid sequence of the heavy (H)-chain is known. Here we report the complete amino-acid sequence of the light (L)-chain of the monoclonal immunoglobulin Kol (IgG1). The polypeptide has an Mr of 22,781, consists of 216 amino acids and due to its structure is of the lambda-type. With the characteristic amino acids threonine, asparagine, threonine, glycine and lysine in positions 101, 114, 116, 154, and 165, respectively the Kol L-chain is of the Mcg isotype. With the proteins Mcg, Mot, Bur, Loc and Mem six myeloma-derived amino-acid sequences of the same isotype are known. The amino-acid sequence of the N-terminal variable part is characteristic of subgroup 1. This contribution completes the primary structure of IgG1 Kol.

Alkylation↗
PubMed1987

Structural relationship of an apolipoprotein (a) phenotype (570 kDa) to plasminogen: homologous kringle domains are linked by carbohydrate-rich regions.

At least six allelic forms of apolipoprotein(a), differing in molecular mass, could be detected by immunoblot analysis. One of these phenotypes with a molecular mass of 570 kDa has been investigated. After reduction and carboxymethylation it was digested with trypsin and the resulting peptides were separated by gel filtration and reverse phase HPLC. The tryptic fragments sequenced comprised a total of 356 amino acids. The N-terminus of apo(a) was highly homologous to the start of the kringle 4 domain from human plasminogen and the majority of the tryptic peptides isolated was also homologous to sequences from this kringle. At least five homologous "kringle 4" domains are present in apolipoprotein(a) whereby one domain occurs more frequently than the others. A carbohydrate-rich peptide was also obtained in high yield. This glycopeptide connects two "kringle 4" domains and contains one N-glycoside within the kringle and six potential O-glycosides in the linking region. From the recovery it can be estimated that this peptide occurs several times within the whole apolipoprotein (a) sequence. The high carbohydrate content is in sharp contrast to that of human plasminogen. Other peptides sequenced indicate that apo (a) also contains domains homologous to the kringle 5 and protease regions of plasminogen. No unique peptides were found. These studies suggest that apolipoprotein (a) could have arisen through duplication of specific regions from the human plasminogen gene. The size heterogeneity of apo (a) might then be explained by differences in the numbers of gene duplications.

Amino Acid Sequence↗
PubMed1985

Thrombin specificity. Selective cleavage of antibody light chains at the joints of variable with joining regions and joining with constant regions.

Selective cleavage of polypeptides by alpha-thrombin can be reasonably predicted [Chang, J.Y. (1985) Eur. J. Biochem. 151,217-224]. This knowledge was applied to the selective cleavage of antibody light chains with the aim of producing intact fragments of both variable region and constant region. (a) Mouse kappa light chains 10K26 and 10K44 from anti-(azobenzene arsonate) antibodies contain 20 Arg/Lys-Xaa bonds. Only two of them, one ProArg-Thr bond located at the joint of the variable region with the joining peptide and one ValLys-Ser bond located near the carboxyl-terminal end of the constant region, were selectively cleaved by alpha-thrombin. The ProArg-Thr bond has a 50% cleavage time of about 10 min under the designated conditions, whereas the ValLys-Ser has a 50% cleavage time approx. 9-10 h. A single selective cleavage at the joining position of the variable region and joining peptide can be achieved by short-time thrombin digestion. Fragments containing intact variable region (1-96) and intact joining peptide-constant region (97-214) obtained from both denatured and native light chains of 10K26 can be separated by gel filtration. (b) lambda light chains from both human and mouse all begin with the GlnProLys-(Ala/Ser) structure (positions 108-111) at their constant regions. This ProLys-Ala/Ser bond is also susceptible to specific thrombin cleavage. Four human lambda chain (KERN, NEI, NEW, VOR) and one mouse lambda chain (RPC20) were shown to be selectively cleaved by thrombin at these ProLys-Ala/Ser bonds. For human lambda chains, the 50% cleavage time at this ProLys-Ala bond was approx. 3-4 h under the designated conditions. Six additional thrombin specific cleavages were also detected within the variable regions of NEI, VOR and RPC-20. (c) Heparin inhibits thrombin cleavage of Arg/Lys-Xaa bonds located near the center of the antibody light chain, but slightly activates thrombin cleavage of those located near the amino or carboxyl-terminal ends of the protein. The significance of these findings is threefold. (a) It demonstrates that selective cleavage of large polypeptides by alpha-thrombin can also be reasonably predicted. (b) It provides a useful method for light chain fragmentation which can greatly facilitate amino acid sequencing of antibodies. (c) It serves to generate fragments containing intact variable regions and constant regions from antibody light chains of human and mouse. Such fragments may be useful for chemical semisynthesis of a human-mouse light chain chimeras.

Amino Acid Sequence↗
PubMed1984

The primary structure of mu-chain-disease protein BOT. Peculiar amino-acid sequence of the N-terminal 42 positions.

The complete primary structure of the mu heavy-chain disease (mu-HCD) protein BOT has been determined. The monomeric HCD-mu-chain consists of 391 amino-acid residues, lacking the VH and mu CH1 domains but including the entire CH2, CH3 and CH4 domains (349 residues). The sequence of the preceding 42 N-terminal residues which we designate as the "pre-C-part" presents no homology to any known variable or constant immunoglobulin sequence, but contains an internal homology of positions 10-19 to positions 20-29. The origin of the "pre-C-part" structure and the deletion of the mu CH1 domain of protein BOT are discussed.

Amino Acid Sequence↗
PubMed1984

[Primary structure of human class II histocompatibility antigens (HLA-D). I. Isolation, purification and characterization of the HLA-D alpha/beta chain complex from a homozygous lymphoblastoid B cell line, H2LCL (HLA-A3,3;B7,7;Dw2, 2;DR2,2;MT1,1;DC1,1;MB1,1].

The complex of alpha and beta chains of HLA-D membrane antigens has been isolated from a lymphoblastoid homozygous B cell line, H2LCL (HLA-A3,3; B7,7; Dw2,2; DR2,2; MT1,1; DC1,1; MB1,1), by an exclusively chemical two-step procedure and characterized by electrophoresis as well as isoelectric focusing in polyacrylamide gel. Cells were gained using long term cultivation in large scale, the crude membrane by differential centrifugation. The proteins of the crude membrane were then solubilized in NP-40, pH 5.0. The first purification step for HLA-D antigens consisted in an ion-exchange chromatography on carboxymethyl cellulose using the solubilization buffer. By this procedure the complex of proteins with relative molecular masses of Mr approximately 34 000 and Mr approximately 29 000 was in a high percentage not bound to the carboxymethyl cellulose. The bound fraction contained the HLA-A, -B and -C antigens and a component with Mr approximately 31 000 corresponding to the well known Ii-fraction. The bound proteins could be recovered from the column by a sodium chloride gradient. The proteins not bound to the carboxymethyl cellulose were precipitated with acetone, dissolved, dialysed against SDS buffer, pH 7.2 and then submitted to the second purification step, the Sephacryl S-300 chromatography. By this procedure the corresponding complex could be further separated from higher and lower molecular proteins. The complex was used as the starting material for the separation of alpha and beta chains. Amino-acid sequences established of the isolated chains have already been communicated.

Amino Acid Sequence↗
PubMed1984

[Primary structure of human class II histocompatibility antigens (HLA-D). II. Separation of alpha from beta-chains of HLA-D from a homozygous lymphoblastoid B cell line, H2LCL (HLA-A3,3;B7,7;Dw2,2;MT1,1;DC1,1;MB1,1), and characterization of the isolated chain].

In a previous communication we described the preparation of the alpha/beta-chain complex of HLA-D membrane antigens from a homozygous lymphoblastoid B cell line, H2LCL (HLA-A3,3; B7,7; Dw2,2; DR2,2; MT1,1; DC1,1; MB1,1). In this paper we present the separation of the alpha- and beta-chains in quantitative scale using hydroxylapatite chromatography with a pH 7.15 phosphate buffer, containing sodium dodecyl sulfate. An alternative isolation procedure is electrophoresis, taking advantage of the different isoelectric points of the alpha- and beta-chains. The relative molecular masses, the isoelectric points and the N-terminal sequences are discussed and compared with the results of other investigators. Remarkable is the polymorphism of both the chains, especially the beta-chains. The importance of the previously described three step purification procedure for the preparation of these antigens for sequence studies has been pointed out.

B-Lymphocytes↗
PubMed1984

[The primary structure of human free secretory component and the arrangement of disulfide bonds].

The amino-acid sequence and the arrangement of the disulfide bonds of the human free secretory component were completely elucidated by the methods of protein chemistry. The free secretory component is a monomeric glycoprotein (Mr approximately 86000), consisting of 558 amino acids with 7 carbohydrate chains bound to asparagine. The protein contains 20 cysteine residues but, as a special feature, no methionine. The polypeptide chain is divided into five regions of internal homology, 104 to 114 amino acids in length. The 20 cysteine residues form 10 disulfide bonds, 9 of which confirm the internal homology by their characteristic arrangement. The free secretory component also shows homology to immunoglobulins in some sections. A computer-supported tertiary structure is proposed for the free secretory component.

Amino Acid Sequence↗